Formation of a ternary complex between thrombin, fibrin monomer, and heparin influences the action of thrombin on its substrates.
The consequences of the combined effects of fibrin II monomer (FnIIm) and heparin (H) on the hydrolysis of peptidyl p-nitroanilide substrates by thrombin (IIa), the cleavage of prothrombin by thrombin and the thrombin-catalyzed release of fibrinopeptides from fibrinogen have been studied at pH 7.4 and I 0.15. The effects of fibrin II monomer and heparin on chromogenic substrate hydrolysis can be described by a hyperbolic mixed inhibition model in which substrate can interact with four possible enzyme species (IIa, IIa.H, IIa.FnIIm, and IIa.FnIIm.H) that arise as a result of random formation of a ternary complex among thrombin, fibrin II monomer, and heparin (Hogg, P. J. and Jackson, C. M. (1990) J. Biol. Chem. 265, 241-247). The formation of the ternary IIa.FnIIm.H complex results in an increase in the Km values of 7.03 +/- 1.17-fold (1.37-9.65 microM) and 1.94 +/- 0.60-fold (38.1-73.9 microM) for H-D-Ile-Pro-Arg-pNA and Cbz-Gly-Pro-Arg-pNA hydrolysis, respectively, and a decrease in the kc values of 0.45 +/- 0.08-fold (49.5-22.3 s-1) and 0.52 +/- 0.05-fold (93.1-48.4 s-1). Fibrin II monomer and heparin in combination also decrease the efficiency (kc/Km) with which thrombin cleaves prothrombin to produce Fragment 1 and Prethrombin 1 by 2.3-fold from 607 +/- 30 to 264 +/- 13 M-1 s-1. In contrast to the effects of fibrin II monomer and heparin on thrombin hydrolysis of chromogenic substrates, its proteolysis of prothrombin and its inactivation by antithrombin III (Hogg, P. J., and Jackson, C. M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3619-3623), these components have no discernible influence on the ability of thrombin to cleave fibrinogen. These observations indicate that the substrate specificity of thrombin is altered when it is bound in a complex with fibrin II monomer and heparin and suggest that the catalytic efficiency of thrombin for its physiological substrates will be affected differentially by these interactions. Such ternary complex formation involving thrombin, fibrin II monomer, and heparin may provide a mechanism for selectively regulating thrombin action.
Hogg
P J
PJ
American Red Cross Blood Services, Southeastern Michigan Region, Detroit, Michigan 48232.
Jackson
C M
CM
D000595:Amino Acid Sequence; D002863:Chromogenic Compounds / Q000378:metabolism; D005338:Fibrin Fibrinogen Degradation Products / Q000378:metabolism / Q000494:pharmacology*; D005340:Fibrinogen / Q000378:metabolism; D005344:Fibrinopeptide A / Q000378:metabolism; D005345:Fibrinopeptide B / Q000378:metabolism; D006493:Heparin / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D011516:Prothrombin / Q000378:metabolism; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism*
D002863:Chromogenic Compounds; D005338:Fibrin Fibrinogen Degradation Products; D046911:Macromolecular Substances; D009842:Oligopeptides; C011468:fibrinmonomer; D005344:Fibrinopeptide A; D005345:Fibrinopeptide B; C040410:carbobenzoxyglycyl-prolyl-arginine-4-nitroanilide; C041319:isoleucyl-prolyl-arginine-4-nitroanilide; D011516:Prothrombin; D005340:Fibrinogen; D006493:Heparin; D013917:Thrombin